The pineal gland is an endocrine organ that is part of our circadian clock system in the brain. Throughout the night, noradrenaline released locally by the sympathetic nervous system stimulates the pineal to broadcast the night hormone, melatonin, to all of the body. We will study the early second-messenger signaling within isolated pinealocytes in response to applied norepinephrine, the longer term availability of adrenergic receptors during prolonged exposure to norepinephrine, and, in pineal slices and whole pineal glands, the modulatory actions of classical neurotransmitters on pinealocyte activity. We will determine the G-protein coupling of melatonin receptors expressed in various expression systems. Finally we will test the hypothesis that N-acetyl serotonin is a candidate for another night hormone in addition to melatonin. Cells will be dissociated from rat pineal glands, placed in cell culture, treated with norepinephrine to mimic night, and studied under the microscope with techniques such as whole-cell gigaseal electrophysiological recording, live-cell photometry of calcium-sensitive dyes and of indicators for second messengers, and HPLC-mass spectroscopy of the three indoleamine hormones. Solvent extracts of whole pineal glands will be analyzed by HPLC-mass spectroscopy for the phosphoinositides and other phospholipids of their membranes. Under- standing the rhythmic secretory mechanisms of the pineal will make an important contribution towards treating sleep disorders, seasonal affective responses to short days, and loss of attention and productivity due to jet lag and shift work.